Neural cultures

ABSTRACT

The invention relates to a method of producing nerve cell-lines a homogeneous population of nerve cells having preselected biochemical/functional characteristics. In addition, the invention also concerns the provision of a homogeneous population of cells which can be selectively made to undergo apoptosis. Finally, the invention also concerns nerve cell-lines provided by the method of the invention.

[0001] The invention relates to a method of producing neural culturesand particularly, but not exclusively, neural cell-lines; and the cellsand cell-lines produced by such a method.

[0002] The invention also relates to human and animal neural cell-lines,and particularly but not exclusively, nerve cell-lines.

[0003] Nerve cells are highly differentiated cells comprising a cellbody, and processes, the latter subdivided into dendrites and axons.Nerve cells vary considerably in shape and size in different parts ofthe body. For example, granule cells from the cerebellum are 5micrometres in diameter whereas the large motor cells of the anteriorhorn of the spinal cord are up to a 120 micrometres in diameter. Inaddition, the axons of nerve cells vary from about a hundred micrometresin length up to 1 metre in length. In addition to this variation inshape and size nerve cells also vary in the nature of the receptorsexpressed on their cell surface and the nature of the neurotransmitterssecreted for the purpose of effecting nerve cell transmission. Thisdifference in biochemistry can be used for the purposes ofclassification. Thus, in simplistic terms, nerve cells may be classifiedas, for example, adrenergic, cholinergic, serotoninergic, dopaminergicetc according to the nature of their neurotransmitters. This biochemicalmode of classification can be further sub divided in order to identify awhole range of nerve cells secreting different neuropeptides that arethought to function as neurotransmitters or neuromodulators such as theneuropeptides beta endorphin, met — enkephalin, somatostatin,luteinizing hormone — releasing hormone, thyrotropin releasing hormone,substance P, neurotensin, angiotensin 1, angiotensin 2, vasoactiveintestinal peptide, neuropeptide Y, calcitonin gene related peptide etc,or alternatively, the amines or amino acids, adrenalin, noradrenaline,octopamine, serotonin, histamine, gamma aminobutyric acid, and taurine.The afore list is not intended to be exhaustive but rather serves toillustrate the nature of the biochemical diversity of nerve cells.

[0004] It is widely acknowledged that it would be irmmenselyadvantageous if it was possible to provide ideally in culture ahomogeneous population of nerve cells and so provide, for example, ahomogeneous population of nerve cells either from a given location inthe central nervous system, or alternatively, a homogeneous populationof nerve cells exhibiting either predetermined morphologicalcharacteristics and/or biochemical characteristics. For instance itwould be highly advantageous if it was possible to provide a homogeneouspopulation of nerve cells which were characterised by either thetransmitter secreted in response to activation or alternatively thereceptor occupied in response to activation. With such a population ofnerve cells it would be possible for research biologists to makesignificant advances in the understanding of the nervous system and forindustrial biologists to manufacture and test drugs, agents or entitieswhich affect the functioning of a given population of nerve cells with aview to developing therapeutically active agents.

[0005] In addition, if it was possible to provide a homogeneouspopulation of nerve cells it would be possible to provide nerve cells ofa given classification for the purpose of transplantation. This would beparticularly appropriate in cases where nerve cell degeneration ordamage had occurred. For example, it is well known that Parkinson'sDisease is related to nerve cell degeneration and a corresponding lackof secretion of dopamine by nerve cells. Thus, if it was possible toprovide a homogeneous population of nerve cells that secrete dopaminethen it would be possible to transplant such nerve cells and thusmitigate or alleviate or even reverse the symptoms of Parkinson'sDisease. Similarly, other forms of dementia which are characterised by aprogressive degeneration of nerve cells could be treated in a similarmanner. Similarly, acute destruction of nervous tissue could be treatedby nerve cell implants comprising a homogeneous population of nervecells and/or the implantation of a selected combination of nerve cellsfrom different homogeneous populations.

[0006] However, the above referred to diversity of nerve cells and alsothe postmitotic nature of nerve cells tend to impose severe restrictionson the number of cells that can be obtained in vitro for investigationand/or transplantation using conventional cell culture techniques. Forthis reason attempts have been made to provide cultures of nerve cellsby cultivating primary tumour tissue or by fusing primary cells withtumour cells. However, tumour cells are irreversibly transformed andhave an ill-defined history. Their use as cell models is thereforehighly questionable and moreover because of the potential tumorigenicityof such tissue they cannot be used for the purpose of transplantation.

[0007] Attempts to provide homogeneous populations of nerve cells havealso been undertaken using carcinogen-induced transformation both invivo and in vitro and also by spontaneous transformation that is to sayby the out growth of cells from primary cultures without any deliberategenetic manipulations.

[0008] However, it has been found that another restriction on theprovision of homogeneous populations of nerve cells concerns the factthat most neural tumours are human glioblastotnas and thus do notconcern the uncontrolled division of functional nerve cells.

[0009] Other workers have transfected neural cells with oncogenes inorder to establish neural cell-lines. Some workers have shown that it ispossible to induce oncogenes into primary neural cells and to obtaincell-lines, however, these cell-lines are not nerve cell-lines. They arenot functioning nerve cells nor are they homogeneous populations ofdefinable nerve cells (1).

[0010] The transfection techniques used in the past have involved theuse of retroviruses because of the ability of such viruses to stablyintegrate into the host cell genome. In addition, transfection has beenundertaken using a temperature sensitive mutant of the DNA virus simianvirus 40 (SV40). The A gene of SV40 encodes the large tumour (T) antigenwhich is required for the initiation and maintenance of transformation.

[0011] Integration of viral genes into host cell genomes requires thatthe host cell undergoes at least one round of DNA synthesis. Ittherefore follows that where integration of a viral gene into a hostcell is required target cells are limited to mitotic neural cells.Transfection techniques have therefore been undertaken on such cells.Although it has been possible to produce cell-lines, that is to say ithas been possible to immortalise the transfected cells, it has not beenpossible to produce immortalised cells with the required degree ofdifferentiation which would render such cells as useful tools forfurther research, study or use. This would seem to be becauseimmortalisation prevents terminal differentiation of nerve cells.Indeed, typically the cells enter crisis and apoptosis ensues. Forexample, when immortalisation of neural cells takes places using SV40 Ta homogeneous population of cells can be cultured, however at a nonpermissive temperature of 39° C. expression of the active viral proteinceases and the cells enter differentiation. However, differentiationdoes not proceed to completion, the cells enter crisis and apoptosisensues.

[0012] In addition, it also widely acknowledged that it is extremelydifficult to provide in culture differentiated neural or nerve cellseither for use in transplantation and/or for use in testing drugs,agents or entities which effect the functioning of a given population ofnerve cells with a view to developing therapeutically active agents. Itis difficult to provide such a culture of nerve cells, especially whereone is trying to provide, largely, a homogeneous population of nervecells, or a heterogeneous population of nerve cells including arelatively small number of phenotypes, because amongst other things, itis very difficult to provide for differentiation of such nerve cells.Typically it is difficult to provide for differentiation of primarynerve cells in culture.

[0013] It is therefore an object of the invention to provide a methodfor producing nerve cell-lines which represent homogeneous populationsof nerve cells which are not only functional but whose character can bereliably defined. In other words it is an object of the invention toprovide a method for producing a stable nerve cell-line which iscommitted to its phenotype. For example, using the invention it ispossible to provide a homogeneous population of functional serotonincells or acetylcholine cells or adrenalin cells etc.

[0014] It is a further object of the invention to provide a non-mitoticcell-line, whose non-mitotic characteristics persist even in thepresence of factors and/or conditions which would normally promotemitosis.

[0015] It is yet a further object of the invention to provide acell-line which survives at low densities.

[0016] It is also an object of the invention to provide a method forproducing nerve cell-lines which can be selectively made to enterapoptosis so that the process of apoptosis can be studied with a view to6aina a greater understanding of the process and also with a view toengineering drugs, agents or entities that affect apoptosis.

[0017] It is yet a further object of the invention to provide for apopulation of nerve cells, homogeneous or otherwise which are fullydifferentiated.

[0018] The method of the invention is based on a startling observation.Using conventional transfection techniques we were able to immortaliseselected neuronal cells. However, as with many other workers, untilrealising the invention, we were unable to provide fully functionaldifferentiated nerve cells. However, when we modified our method forproducing cell-lines we found that we were able to induce fulldifferentiation of our nerve cells when they were exposed, followingtransfection and immortalisation, to predetermined conditions. Theseconditions involved exposing the cells to either the environment fromwhich they came and particularly, but not exclusively, the mitoticenvironment from which they came or to conditions which mimicked theenvironment from which they came and thus provided for an artificialimitation of the environment from which they came.

[0019] Our observation has also enabled us to produce an in vitroculture of nerve cells which have not been immortalised. In thisinstance, primary nerve tissue is first encouraged to replicate byexposure to a replicating agent (8 and 9) and is then encouraged todifferentiate by exposing the cell culture to the aforementionedenvironment from which said primary tissue came or to conditions whichmimic said environment.

[0020] By the term, the environment from which they came, we mean aregion of the central nervous system, and more preferably a region ofthe central nervous system at, adjacent, or functionally related to thenatural location in the central nervous system of the cultured cells. Wefavour a mitotic environment therefore we favour a region from thecentral nervous svstem which is mitotically active and more preferablywe favour a region from the central nervous system at, adjacent, orfunctionally related to the natural location in the central nervoussystems of the cultured cells.

[0021] It would seem that having to expose the cells to the environmentfrom which they were derived means that cells of that environmentsecrete agents, such as for example cytokines, growth factors,transmitters etc or perhaps such cells comprise removable cell surfacebased factors, which can elicit a differentiation response.

[0022] In addition, we have found that it is possible to use tissue andcells from different species in order to work the invention. Forexample, it is possible to culture human nerve cells and expose suchhuman nerve cells to said environment or said artificial environmentwhich is derived from rat central nervous systemn. Conversely, it ispossible to culture rat nerve cells and expose said nerve cells to anenvironment or artificial environment which is human derived.

[0023] It would therefore seem that agents which elicit neuronaldifferentiation of the invention are agents which can elicit theireffects cross species. That is to say these agents are biologicallyactive in at least both rat and human systems and are therefore likelyto be of the same or similar structure.

[0024] Thus we have found that modifying our method such thantransfected cells or cultured cells are exposed to the conditions of theoriginal environment at least from which the first culture cell camebrings about differentiation. We are unclear as to the nature of thefactors involved at this stage.

[0025] Further, when using transfected cells we prefer to employ amethod which includes the provision of a switch which enables us tocontrol immortalisation and apoptosis. Using our method we have foundthat cultured nerve cells do not spontaneously undergo apoptosis sofrustratingly characteristic of previously cultured neural cell-lines,but rather we can selectively control whether cell-lines remainimmortalised or enter apoptosis.

[0026] In addition, we have also found that our cell-lines whendifferentiated, are committed to their phenotype and thus retain theirphenotypic characteristics even when the environment from which theycame is removed and/or they are exposed to factors such as foetal calfserum. Further, we have also found that our cell-lines do not exhibitmitosis, again, even under conditions which would promote mitosis, and,what is more, our cell-lines are able to survive at low densities.

[0027] According to a first aspect of the invention there is thereforeprovided a method for producing large populations of neural cells whichmethod involves:

[0028] a) enhancing the replication of a first undifferentiated neuralcell, or neural cell precursor cell, or precursor stem cell,

[0029] b) exposing said replicated neural cells either to an environmentfrom which said first neural cell came, or to an environment whichmimics said environment; and

[0030] c) allowing differentiation of said cells to produce fullydifferentiated active neural cells.

[0031] It is apparent from the above that using the method of theinvention one is able to culture and/or immortalise a neural cellprecursor cell and thus produce a homogeneous population of cells.However, successful differentiation is effected by exposing the cells toeither the environment from which the first nerve cell came oralternatively to an environment which mimics that environment. In thisway, it is possible to produce a homogeneous population of fullydifferentiated active neural cells.

[0032] In a first embodiment of the invention the environment from whichthe first nerve cell came is any region of the central nervous system,however, more preferably, said environment is an environment at,adjacent, or functionally related to the natural location in the centralnervous system from which the cultured cells derive. The term, anenvironment which mimics said environment, is also to be construedaccordingly.

[0033] More preferably still, said environment is a mitotic environment,that is to say, it comprises cells undergoing mitosis. It would seemthat in this instance the agent(s) which elicit the differentiationprocess are being released or expressed and somehow affectingdifferentiation by cells within the mitotic cells environment.

[0034] Preferably, said nerve cells and tissue from said natural orartificial environment is derived from a single species. However,alternatively, said nerve cells and said tissue may be derived fromdifferent species. For example, said nerve cells may be derived fromfoetal human tissue whereas said environment and more specifically saidtissue of said environment may be derived from an another animalspecies. such as rat, mice, monkeys etc.

[0035] In a preferred embodiment of the invention immortalisation isachieved by using conventional transfection techniques and preferablythe transfection involves the incorporation into the cell aenome of anoncogene which oncogene favours the establishment of cell division wellbeyond the normal level encountered when a cell is not transduced withan oncogene, in other words the oncogene immortalises the cell.

[0036] Alternatively, immortalisation maybe effected using physical orchemical means. For example, immortalisation maybe effected by exposingsaid cell to radiation or chemicals (2) which are known to promote celldivision well beyond the normal level encountered when a cell is notexposed to said physical and chemical means.

[0037] Ideally transfection is undertaken using a virally derivedoncooene such as a myc, src, ras, SV40T, or a retroviral constructincluding any of the aforementioned oncogenes and/or any humanoncogenes. A retroviral construct is favoured because of its ability tostably integrate into the host cell genome.

[0038] In a first preferred embodiment of the invention theimmortalising agent includes or has associated therewith a control meanswhereby activation of the control means terminates immortalisation and.causes the cell to enter apoptosis.

[0039] It is preferred that immortalisation of said cell with animmortalising agent takes place ideally during the last division beforemigration from the proliferative zone and the onset of terminaldifferentiation. This is because the likelihood of producing a cell-linehaving a single set of functional characteristics is increased.Immortalisation prior to this preferred time can be undertaken but thelikelihood of the precursor cells adopting several different phenotypesafter differentiation is increased.

[0040] In a preferred embodiment of the invention the control means isresponsive to culture or environmental conditions such as temperature,pH or ionic concentrations. For example, in a preferred embodiment theimmortalising agent is temperature sensitive and the control is thusrepresented by a temperature sensitive switch so that at, about, orbelow a first given temperature the immortalisation agent is activatedso as to immortalise the selected nerve type, but at, about, or above asecond temperature the immortalising agent is deactivated and in thisinstance immortalisation terminates and apoptosis is allowed to proceed.The immortalisation agent and the control means may comprise, forexample, a single entity such as a temperature sensitive oncogene.Alternatively, the immortalisation agent and the control means may betwo independent entities but in either case ideally theactivation/deactivation of the control means has a reciprocal effect onthe immortalisation agent. For example, when the control means isactivated the immorntalisation agent is deactivated. Conversely when thecontrol means is deactivated the immortalisation agent is activated.This ability of the control means to deactivate the immortalisationagent is a means of terminating immortalisation such that apoptosis cantake place.

[0041] Exposing said cells to the original environment can involvetransplanting said homogeneous population of cells back into the centralnervous system or more preferably a location in the central nervoussystem at, adjacent or functionally related to the original enviromnentof the first cell or alternatively, and more preferably, simplyextracting a population of cells from said central nervous system orsaid orginal environment and placing said extracted population in closeproximity to said homogeneous population of cells.

[0042] Ideally, said chosen environment comprises mitotically activecells.

[0043] In the instance where said cell is exposed to an extractedpopulation of cells then ideally said extracted cells are plated onto asubstrate and allowed to reach confluence either before being placed incontact with said homogeneous population of cells or whilst in contactwith said homogeneous population of cells. Alternatively, said extractedpopulation of cells are grown to confluence and medium from saidpopulation is added to said homogeneous population of cells in order tobring about differentiation.

[0044] Preferably, said homogeneous population of cells are also exposedto one or more growth factors such as fibroblast growth factor and/orepidermal growth factor.

[0045] It will be apparent from the above that the nature of thehomogeneous population of cells will be determined by the nature of theundifferentiated nerve cell or nerve cell precursor cell. Thus using themethod of the invention it will be possible to produce cell-lines ofdifferent nerve cells whose function and properties will be determinedby the nature of the undifferentiated nerve cells or nerve cellprecursor cells. Thus the invention has wide ranging application in thatthe invention provides a method whereby a whole range of homogeneouspopulations of nerve cells can be grown in culture. This is obviouslysignificant for neurobiologists both from a research point of view andfrom a technical point of view.

[0046] Preferably the immortalising agent is, what is typically referredto as, a soft oncogene such as a SV40 viral oncogene and morepreferably, in the instance where a control means is preferred theoncogene is the SV40 T antigen which is permissive, that is to say theviral gene active product is expressed, at 33° C. and non-permissive,that is to say the viral gene active product is not expressed, at 39°C., thus cells immortalised using this agent are temperature sensitivefor apoptosis.

[0047] Uniquely, our cells, when transformed using SV40 T antigen andexposed to an environment, natural or artificial, which promotesdifferentiation, survived crisis — a condition which is typicallyfollowed by apoptosis.

[0048] It would seem that the said environment also provides for therelease of substances or somehow effects the cells to enable them tosurvive apoptosis.

[0049] In yet a further preferred embodiment of the invention saidcell-line includes a safety feature which allows for selective disablingor destruction of said cell-line. This safety feature is of advantagewhere the cell-line is to be used for the purpose of transplantation oris otherwise, whether it be permanent or temporary, attached to,administered to, or stored in, an individual. This safety feature allowsthe cell-line to be selectively disabled, and by this we mean renderedharmless, or destroyed, in instances where the cell-line is thoughtlikely to, or is shown to, have the potential to become tumorclgenic invivo, or is thought to be in any way harmful to an individual.

[0050] Our copending patent application GB 9421236.1 teaches how avector can be produced which provides for co-expression of a safetyfeature in the form of a gene which may or may not be linked to theimmortalising oncogene.

[0051] According to a further aspect of the invention there is providedcells and/or cell-lines produced in accordance with the method of theinvention. Accordingly there is provided at least one homogeneouspopulation of immortalised cells which can be made to fullydifferentiate so as to provide a homogeneous population of fullydifferentiated nerve cells; and/or alternatively, there is provided atleast one homogeneous population of immortalised cells provided withmeans to terminate immortalisation and activate apoptosis.

[0052] According to a yet further aspect of the invention there isprovided cells and/or cell-lines produced in accordance with a method ofthe invention which, when differentiated, retain their phenotypiccharacteristics and/or are non-mitotic and/or survive at low densities.

[0053] An embodiment of the invention will now be described by way ofexample only and for the purpose of example only with specific referenceto serotonin secreting functional nerve cells.

[0054] The following is exemplified by a table and a number of figureswherein;

[0055] 1. Table 1 is a summary of experiments performed with clone 1immortal nerve cell-line.

[0056] 2. FIG. 1 shows the current-voltage relationship at 30 mM Ba ofclone 1 cells one to two weeks after differentiation.

[0057] 3. FIG. 2 shows time course of VDCC effects of toxin applicationon clone 1 nerve cells; and

[0058] 4. FIG. 3 shows current-voltage relationship for clone 1 cells.

[0059] Immortalisation of Cells

[0060] Rat embryos at days 12-13 of gestation were dissected, and thepresumptive raphe nuclei region comprising the ventral medialrhombencephalon and medulla oblongata were removed. After dissociationby gentle trituration in medium (Ham's F12/Dulbecco's modified Eagle'smedium (50/50 v/v) supplemented with L-glutamine (2 mM),penicillin:streptomycin (100 IU/ml: 10 μg/ml) and modified stocksolution [3,4] containing 5 ng/ml basic fibroblast growth factor [5](all supplied by Sigma), cells were plated ontopoly-L-lysine/gelatin-coated 162 cm² tissue culture flasks (Costar UKLtd) at a density of 5×10 cells/ml, 20 ml per flask. Once the cells hadadhered, retroviral particles comprising a construct (tsA58)incorporating a temperature sensitive form of the simian viruslaroe-tumour antigen (ts)SV40-T and a resistance marker to geneticin,G418′(kindly donated bv Dr P Jat, Ludwig Institute, Middlesex Hospital,London. Also available on deposit, details to be provided)[6], wereadded to the medium together with 0.8 μg/ml polybrene. The viral titrewas adjusted to give a low transduction efficient of 0.0002%, producingan average of 20 colonies per flask. After 1 h, the culture medium wasreplaced with fresh medium. Cultures were maintained at 33° C., thepermissive temperature for the active form of the SV40-T, oncogeneproduct. Five days after transduction, geneticin was added to theculture medium (0.4 mg/ml) for a further 8 days to eradicate cells whichhad not incorporated the retroviral vector.

[0061] Between 14 and 20 days after transduction, individual colonies ofreplicating, cells were identifiable. Clones were selected on the basisof being well separated from other replicating colonies, their circularshape and their morphology. Individual clones were picked and expandedup to near confluence in a 75 cm² flask, ie approximately 23 divisionsof a single precursor, prior to freezing down of cell aliquots. Aliquotswere also plated onto poly-L-lysine/gelatin-coated 12-well plates foranalysis of potential differentiation characteristics.

[0062] Alternatively, rat embryos, as aforementioned, were treated so asto provide cells in tissue culture and these cells were then exposed toa replicative agent as described in references 8 and 9 prior toundergoing differentiation as described below.

[0063] Cell Differentiation

[0064] Cells were maintained in the medium constituents as forreplication above and at the permissive temperature of 33° C., but thehomogeneous population of nerve cells, now referred to as RAPHE CLONE 1CELLS, were cocultured in the bottom of a well with primary RAPHE CELLS(prepared as above but without the steps subsequent to transfection) asa non-confluent cell layer plated on PTFE inserts (Corning). Both theimmortalised and the primary RAPHE cells replicated ,until the primaryRAPHE cells became confluent. At this point the immortalised cellsexhibited a much reduced replication rate. The primary RAPHE cells wereremoved by removing the insert and the immortalised cells began toexhibit a significant degree of differentiation. For example,5-hydroxytryptamine was now synthesized without the requirement forprecursor loading, that is native 5-hydroxytryptamine was nowdemonstrable. Morphological differentiation was much more complex, inthat many tapering dendrites, branching often were visualised. Inaddition, the cells developed several ion channels, including inparticular N-type calcium channels. Little or nor apoptosis was seen atthe permissive temperature, and the cells were refractory to the glialdifferentiation-inducing effects of serum.

[0065] Cell Apoptosis

[0066] The above referred to homogeneous population of raphe clone 1cells can be caused to enter apoptosis using any of the following fourmethods.

[0067] 1. Temperature was raised to non-permissive value (39° C.) for upto 72 h, in the presence or absence of fibroblast growth factor orepidermal growth factor. Neural cells developed the, ability to take up5-hydroxytryptophan (5HTP, the precursor of 5HT), 5HT itself, and todecarboxylate 5-hydroxytryptophan to 5HT. No native 5HT was detectable.The 5HT derived from 5HTP was released, although the mechanism of suchrelease is not known. Weak neurofilament and neurone-specificenolase-like immunoreactivity was demonstrable. Morpholouicaldifferentiation was limited to development of three or foursingle-branching neurites. The cells also appeared to undergo extensiveapoptosis, such that after three days fewer than 10% remain. Theremaining cells were probably neuronal.

[0068] 2. Temperature was raised to non-permnissive value (39° C.) forup to 72 h, in the presence of cyclic AMP plus fibroblast growth factor.Although the parameters of differentiation described above are basicallysimilar after incubation of the cells with cyclic AMP, there was anincrease in the extent of fibre development. The cells were probablyneuronal.

[0069] 3. Temperature was raised to non-permissive value (39° C.) for upto 96 h, in the presence of retinoic acid (10μM) plus fibroblast growthfactor. Cell survival was greatly enhanced, but cells failed to developthe 5HT parameters described above. In addition, neurone-specificenolase staining was much reduced, while conversely glial fibrillaryacidic protein immunoreactivity was now found in many but not all thecells. They had taken on a flattened morphology, and no longer exhibitedfibrous extensions. The cells were probably glial.

[0070] 4. Temperature was raised to non-permissive value (39° C.) for upto 96 h, in the presence of 5% foetal bovine serum plus 5%heat-inactivated horse serum plus fibroblast growth factor. Cellsurvival was greater even than after retinoic acid, and the cells lostthe 5HT parameters described above. In addition, neurone-specificenolase staining was dramatically reduced while glial fibrillary acidicprotein immunoreactivity was now found in many more cells. The cellstook on a flattened morphology, and no longer exhibited fibrousextensions. The cells were probably glial.

[0071] We believe that the cells can be made to undergo apoptosis whenthey reach confluence.

[0072] Differentiation Conditions

[0073] Mesencephalic and medullary raphe neural cells from the E12-E13rat embryo (E1=day of conception) were irnmortalised using atemperature-sensitive oncogene as described earlier (Stringer et al.,1994). Under permissive conditions, ie in the presence of 5 ng/ml offibroblast growth factor (FGF)(Sigma, product no. F3391) and at 33° C.,the immortalised raphe precursor cells replicate. In one clone(921203-6), which possessed all of the characteristics of the clone921202-6 described in Stringer et al, (1994), shifting the temperatureto 39° C., but maintaining all other conditions as before, caused theprecursors to develop some of the characteristics of serotoninergic(5HT) neurones, such as neurone-specific enolase- (NSE) andneurofilament- (NF) immunoreactivity, a phase-bright morphology with twoor three short bifurcating processes, the ability to take up serotoninvia a low-affinity carrier (K, =56μM) and to decarboxylate5-hydroxytryptophan (5HTP) to serotonin. Tryptophin hydroxylaseactivity, however, was not demonstrable, and the cells failed tosynthesize serotonin from tryptophan. No calcium channels weredemonstrable using patch clamp analysis.

[0074] Growing clone 921203-6 raphe precursors tin the presence ofprimary cells dissected from the same ventromedial regions of themesencephalon and medulla oblongata from which the clone was originallyderived leads to an enhanced differentiation of the clone, provided amitotic environment is maintained. To establish such conditions, theventromedial mesencephalon and medulla oblongata were dissected from theE12-E13 rat embryo and plated onto poly-L-lysine coated inserts (PTFEmembrane, pore size 0.4 μm, Corning, product no. 25204-6), approximatelyone mesencephalon/medulla oblongata per insert. The primary cells wereincubated under exactly the same replication conditions as those usedfor obtaining replication in the immortalised precursors ie with 5 ng/mlFGF, and at 33° C. After several days, the density of the primary cellsapproached confluence. At this time, cells from raphe clone 921203-6were plated at low density onto a 6-well plate (previously coated withgelatin and poly-L-lysine) and, after confirmation of their attachmentto the substrate, the primary cell-containing inserts were placed in thesame wells, together with their conditioned medium. No direct contactbetween primary and clonal cells was possible; diffusible factors in thecommon medium could have effects on both sets of cells, clonal andprimary cells alike, but the effects on the former are undoubtedlydirect. Incubation conditions were maintained exactly as before, ie at33° C., with FGF. After 2-3 days, the immbrtalised precursors developeda more highly differentiated morphology, with two to three lona,tapering and branching processes (presumably dendrites) and a largerphase-bright soma. Immunocytochemical analysis of the clonal cells atthis point demonstrated NSE-, NF- and serotonin-immunoreativity, thelast, even in the absence of loading with 5HT, 5HTP or tryptophan.Calcium channels were now demonstrable, and included presumed N-type,and non-T, non-P type channels also. Once the inserts became confluent,both the primary and the immortalised cells started to exhibit signs ofstress and death. Serum failed to prevent this. However, removal of theinsert and/or the conditioned medium completely prevented the cellstress and death. Despite the loss of the conditions medium, theimmortalised cells continued to display all the parameters describedabove of the mature 5HT neurones.

[0075] Using the mid-line region of the capital E12-E13 rat spinal cordas the source of primary tissue full differentiation of the clonalcells.

[0076] Inclusion of foetal calf and heat-inactivated horse serum (bothat 5%) in the culture medium had no apparent effect on thedifferentiated clonal serotonin neurones. By contrast, adding sera tothe same cells undergoing the rudimentary differentiation elicited viathe temperature-shift method caused them to lose all their neuronalcharacteristics and adopt instead an astrocytic phenotype.

[0077] Counts were made daily of the number of immortalised raphe neuralprecursors/differentiated serotonin neurones. Although the cellscontinued to replicate for the first two to three days, as soon as theonset of differentiation became morphologically apparent, replicationceased, even though the cells were still held at 33° C. Removal of theinsert after differentiation had been induced led to no increase in cellnumber; furthermore, no evidence of mitotic bodies was apparent. On theother hand, removal of the insert before differentiation had begunallowed the cells to continue dividing.

[0078] In summary, returning immortalised raphe neural precursor cellsto the mitotic environment from which they originally came leads to amuch more extensive differentiation than previously described methodscan provide. The effect is directly on the clonal precursor cellsthemselves, and is mediated neither via other cell types nor viacell-cell contact. In addition, such differentiation can take place inthe presence of a continuing replicative drive, and leads rapidly to acommitment to the chosen phenotype (eg a full-blown serotonin neurone),which is maintained even in the presence of factors which normally causean alternative phenotype (eg astrocytic) to be expressed. Removal of theconditioning factors does not cause the cells to change from their nowcommitted phenotype. It is likely that soluble factors present in themitotic primary cell-conditioned medium are responsible for inducingsuch differentiation, and may be related to the recently describedN-terminal cleavage product of sonic hedgehog that is known to inducethe differentiation of brainstem and spinal cord precursors to become,respectively, dopaminercgic neurones (Hynes et al, Neuron 15(1995)35-44and cholinergic motoneurones (Roelink et al, Cell(1995)445-455).

[0079] Provision of Nerve Cell-lines includina at least one selectivelvcontrollable safety feature

[0080] Another preferred embodiment of the invention concerns thepreparation of homogeneous populations of cells by retroviraltransduction, but also incorporating a safety feature which enables thecells to be selectively disabled and/or destroyed if needs be. Thiswould be seen as advantageous when such cell-lines are used fortransplantation into patients to alleviate the symptoms ofneurogenerative disorders.

[0081] The safety feature would allow the transplant to be selectivelydestroyed in, for instance, situations where the transplanted materialmay become tumourigenic in vivo and/or situations where the transplantedmaterial becomes harmful in any other way. Ways in which this could bedone are numerous and well known to the man skilled in the art. Forexample, the cell-line may be transfected with a gene which whenactivated acts, either directly or indirectly, to bring about disablingor destruction of the transplant. Examples of such genes are well knownto those skilled in the art and will not be described herein in greaterdetail.

[0082] In a preferred embodiment of the invention a safety featuremay becoupled to the transforming oncogene such that coexpression of the twocorresponding cell products occurs. This means that in instances wherethe oncogene would be activated so too would the safety feature and thusthe dangers associated with the tumourigenic nature of the oncogenewould be overcome. Coexpression could be achieved in a number of waysfor example, the safety gene could be placed downstream of theimmortalising gene and next to but 3′ to for example a poliovirusderived internal ribosomal entrv site sequence (IRES). In this way thesame promoter/enhancer(s) controlling the transcription of theimmortalising gene would, equally, control the transcription of thesafety feature. This is because they would be transcribed as onecomplete unit, including the IRES sequence which would sit in betweenthem. The IRES sequence allows the translation of sequences downstreamof it which code for a separate protein from the sequence 5′ of it. Theability to provide such a vector, once given the idea, is well withinthe range of expertise of a man skilled in the art.

[0083] Experiments to show functional characteristics of thedifferentiated nerve cell-lines

[0084] Functional ion channels

[0085] Table 1 shows the functional activity of clone 1 immortalisednerve cells under varying neurophysiological conditions.

[0086] Twelve different cells were examined either 2 or 4 weeks afterdifferentiation to a fully differentiated nerve cell was complete. Usingconventional patch clamp techniques the conductivity of ion channelswithin the nerve cells examined was determined at either 5 mM Ca or 30mM Ba. At 5 mM Ca cells 2 and 7 showed a conductivity of less than 50pA. Cells 8 and 9 showed a conductivity of greater than a 100 pA. Theseresults indicate that clone 1 included functional nerve cells. At 30 mMBa cells 8 and 9 showed functional ion channels having a conductivitygreater than 200 pA. Cells 11 and 12 also showed conductivity underthese conditions. A weak signal of less than 50 pA was shown for cell 11and a stronger signal of greater than 200 pA was shown for cell 12.

[0087] Exposure of cells from clone 1 to toxins known to interfere withcalcium ion channel conductivity affected the conductivity of clone 1functional nerve cells. Specifically,at 1 mM w-CgTxGVIA, a toxin knownto block N-type calcium channels, cell 8 was 100% affected. As a lowerconcentration of 100 nM cell 9 was 70% affected. At 1 mM cell 12 wasalso 70% affected. These results indicate that factors whichspecifically affect nerve cell conductivity affected the differentiatednerve cells in clone 1 and thus indicate and these differentiated nervecells were fully functional nerve cells expressing phenotypiccharacteristics and more specifically nerve cells possessing N-typecalcium channels.

[0088] Use of the toxin w-Aga IVA, is a toxin known to block P-typecalcium channels, was less successful at a concentration of 50 nM cell 9was insensitive.

[0089] Referrinc now to FIG. 1 current voltage data is available forclone 1 cells. A range of voltages between −85 and 50 mv were applied tothe cells of the invention. Simultaneously the response of said cellswas monitored by recording current flow. Voltages above restingpotential elicited current flow and thus opening of nerve cell ionchannels. A depolarising potential was observed at approximately minus50 mv. This depolarising potential resulted in a generation of an actionpotential indicating that the cells are fully functional. The cells wereinactive at approximately 10 mV.

[0090]FIG. 2 shows a time course of voltage dependent channel conductionand the effects of toxin application on this conductivity. Over a periodof approximately 5 minutes the application of w-CgTxGVIA resulted in amarked reduction in nerve cell conductivity. After a 10 minute intervala second toxin was added and the current remained at approximately 70pA. The current voltage relationship is shown towards the bottom of FIG.2 where it can be seen that the addition w-CgTxGVIA at a concentrationof a 100 nM significantly affected the conductivity of the nerve cellion channels. The addition of w-AgaIVA also affected nerve cellconductivity but much less markedly.

[0091] Finally FIG. 3 shows the current voltage relationship at 30 mM BaCl₂ solution for clone 1 cells. At a depolarising potential of −50 mVnerve cell ion channels are opened and current in the order of −350 pAflows.

[0092] We also have data showing that our ells include fully functionalvoltage dependant potassium channels which are blockable usingconventional physiological tools, (data not shown).

[0093] The above data indicates that the nerve cell clones of theinvention can be made to fully differentiate and thus exhibit phenotypiccharacteristics of a fully functional and thus fully differentiatednerve cell.

[0094] References

[0095] 1. White L A and Whittemore S R., Immortalisation of RapheNeurons: an Approach to Neuronal Function in vitro and in vivo, Journalof Chemical Neuroanatomy, Vol 5:327-330 (1992).

[0096] 2. Stampfer MR, Bartley JC 1985. Induction of transformation andcontinuous cells lines from normal mammary epithelial cells afterexposure to benzo[a]pyrene. Proc Natl Acad Sci USA 82:2394-2398.

[0097] 3. Bottenstein, J E and Sato G H., Growth of a rat neuroblastomacellline in serum-free supplemented medium, Proc. Natl. Acad. Sci.,76(1979) 514-517.

[0098] 4. Romijn H J., Mud M T., Habets, A.M.M.C. and Wolters P S., Aquantitative electron microscopic study of synapse formation indissociated fetal cerebral cortex in vitro, J Nelirophysiol, 40(1981)1132-1150.

[0099] 5. Murphy M, Drago J and Bartlett P F. Fibroblast growth factordirectly stimulates the proliferation and differentiation of neuralprecursor cells in vitro, J Neurosci Res., 25(1990) 463-475.

[0100] 6. Jat P S. and Sharp P A., Cell-lines established by atemperature-sensitive simian virus 40 large-T-antigen gene are growthrestricted at the non-permissive temperature, Mol. Cell Biol, 9(1989)1672-1681.

[0101] 7. Stringer B. M. J., et al., Raphe neural cell immortalised witha temperature-sensitive oncogene, Developmental Brain Research79:267-274, 1974.

[0102] 8. Reynolds B. A. aInd Weiff S. Generation of neurons andastrocytes from isolated cells of the adult mammalian central nervoussystem. Sci. 255:1707-1710, 1992.

[0103] 9. Mayer E., Dunnett F. B., and Fawcett J. W. Mitogenic effect ofbasic fibroblast growth factor on embryonic mesencephalic domapinergicneurone precursors. Dev Brain Res 72:253-258, 1993.

1. A method for producing large populations of netural cells whichmethod comprises undertaking the following steps in the following order;a) enhancing the replication of a first undifferentiated neutral cell,or neural cell precursor cell, or precursor stem cell, b) exposing saidreplicated neural cells either to a region, or part thereof, of thenervous system, or an extract thereof including homologues and analoguesthereof, from which said first neural cell came; and c) allowingdifferentiation of said cells to produce fully differentiated activeneural cells.
 2. A method according to claim 1 wherein the enviornmentfrom which the first nerve cell came is any region of the centralnervous system.
 3. A method according to claim 2 wherein saidenvironment is an environment at adjacent, or functionally related tothe natural location in the central nervous system from which the firstundifferentiated nerve cell is derived.
 4. A method according to claim 3wherein said environment is a mitotic enviornment.
 5. A method accordingto any preceding claim wherein said nerve cells are exposed to a solubleextract from said environment.
 6. A method according to any precedingclaim wherein said enviornment is from the same species as said firstundifferentiated nerve cell.
 7. A method according to claims 1 to 5wherein said environment is from a different species to that of saidfirst undifferentiated nerve cell.
 8. A method according to anypreceding claim wherein enhancing the replication is provided by use ofa replication agent such as a growth factor.
 9. A method according toclaims 1 to 7 wherein enhancing replication is provided by animmoraldising agent.
 10. A method according to claim 9 wherein saidagent is an oncogene.
 11. A method according to claim 10 wherein saidoncogene includes, or has associated therewith, a control means.
 12. Amethod according to claim 11 wherein said control means is responsive toculture or environmental conditions.
 13. A method according to claim 12wherein said control means is responsive to temperature.
 14. A methodaccording to claim 13 wherein said oncogene is SV40T.
 15. A methodaccording to any preceding claim wherein said enviornment comprises anextract of cells from a region at, adjacent, or functionally related tothe original region from which the first undifferentiated nerve cell isdervied.
 16. A method according to claims 1 to 14 wherein saidenvironment comprises a soluble extract taken from a population of cellsphysiologically located at a region at, adjacent, or functionallyrelated to the region from which the first undifferentiated nerve cellis derived.
 17. A method according to any preceding claim wherein saidhomogenous population of cells are exposed to at least one growthfactor.
 18. A method according to any preceding claim which furtherincludes transforming said first undifferentiated nerve cell with asafety feature gene which is either constitutive or can be selectivelyactivated so as to enable, in either case, selective disabling ordestruction of saie cell-line.
 19. Use of a nerve cell-line, whichcomprises a first undifferentiated nerve cell or nerve cell precursorcell that has been immortalised with an immortalising agent whichincludes or has associated therewith a control means whereby theimmortalising agent can be selectively activited/deactivated, as a modelfor investigating apotosis whereby following culuring of saidimmortalised nerve cell so as to provide a homogeneous population ofnerve cells prior to confluence said control means can be activated soas to remove the functionsl effect of the immortalising agent and sobring about cell apotosis.
 20. Cell-lines produced in accordance withthe method of the claims 1-18.
 21. A nerve cell-line according to claims1-18 committed to a fully differentiated phenotype.
 22. A non-mitoticnerve cell-line according to claims 1-18.
 23. A nerve cell-line thatsurvives at low densities according to claims 1-18.